Hypothalamic CRF of the rat and the pig are being purified in collaboration with Dr. A. V. Schally, V.A. Hospital, New Orleans. The activity of CRF is assessed by the release of ACTH from the pituitary tissue of the rat, in vivo and in vitro. ACTH is measured by radioimmunoassay and by bioassay. Partial purification of CRF has been achieved by molecular sieving, ion exchange and counter-current distribution. Further separation procedures based on partition and ionic properties will be applied to purify the material to homogeneity. The purified CRF will be subjected to standard methods to determine amino acid composition and sequence. When pure CRF is available, an immunoassay will be developed. The reason for the relative insensitivity of the neonatal rat to stress is being investigated, with the aim of possibly developing an in vivo assay for CRF. Work is continuing on the kinetics of distribution of labelled ACTH in the rat, and on the characterization of the blood proteins with which ACTH is associated.